The invention relates to nucleoside inhibitors of HCV replicon RNA replication. In particular, the invention is concerned with the use of pyrimidine nucleoside compounds as inhibitors of subgenomic HCV RNA replication and pharmaceutical compositions containing such compounds.
Hepatitis C virus is the leading cause of chronic liver disease throughout the world. (Boyer, N. et al. J. Hepatol. 2000 32:98-112). Patients infected with HCV are at risk of developing cirrhosis of the liver and subsequent hepatocellular carcinoma and hence HCV is the major indication for liver transplantation.
HCV has been classified as a member of the virus family Flaviviridae that includes the genera flaviviruses, pestiviruses, and hapaceiviruses which includes hepatitis C viruses (Rice, C. M., Flaviviridae: The viruses and their replication, in: Fields Virology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 30, 931-959, 1996). HCV is an enveloped virus containing a positive-sense single-stranded RNA genome of approximately 9.4 kb. The viral genome consists of a 5′-untranslated region (UTR), a long open reading frame encoding a polyprotein precursor of approximately 3011 amino acids, and a short 3′ UTR. The 5′ UTR is the most highly conserved part of the HCV genome and is important for the initiation and control of polyprotein translation.
Genetic analysis of HCV has identified six main genotypes which diverge by over 30% of the DNA sequence. More than 30 subtypes have been distinguished. In the US approximately 70% of infected individuals have Type 1a and 1b infection. Type 1b is the most prevalent subtype in Asia. (X. Forns and J. Bukh, Clinics in Liver Disease 1999 3:693-716; J. Bukh et al., Semin. Liv. Dis. 1995 15:41-63).
Unfortunately Type 1 infections are more resistant to therapy than either type 2 or 3 genotypes (N. N. Zein, Clin. Microbiol. Rev., 2000 13:223-235).
Viral structural proteins include a nucleocapsid core protein (C) and two envelope glycoproteins, E1 and E2. HCV also encodes two proteases, a zinc-dependent metalloproteinase encoded by the NS2-NS3 region and a serine protease encoded in the NS3 region. These proteases are required for cleavage of specific regions of the precursor polyprotein into mature peptides. The carboxyl half of nonstructural protein 5, NS5B, contains the RNA-dependent RNA polymerase. The function of the remaining nonstructural proteins, NS4A and NS4B, and that of NS5A (the amino-terminal half of nonstructural protein 5) remain unknown. It is believed that most of the non-structural proteins encoded by the HCV RNA genome are involved in RNA replication
Currently there are a limited number of approved therapies are currently available for the treatment of HCV infection. New and existing therapeutic approaches to treating HCV and inhibition of HCV NS5B polymerase have been reviewed: R. G. Gish, Sem. Liver. Dis., 1999 19:5; Di Besceglie, A. M. and Bacon, B. R., Scientific American, October: 1999 80-85; G. Lake-Bakaar, Current and Future Therapy for Chronic Hepatitis C Virus Liver Disease, Curr. Drug Targ. Infect Dis. 2003 3(3):247-253; P. Hoffmann et al., Recent patents on experimental therapy for hepatitis C virus infection (1999-2002), Exp. Opin. Ther. Patents 2003 13(11):1707-1723; M. P. Walker et al., Promising Candidates for the treatment of chronic hepatitis C, Exp. Opin. investing. Drugs 2003 12(8):1269-1280; S.-L. Tan et al., Hepatitis C Therapeutics: Current Status and Emerging Strategies, Nature Rev. Drug Discov. 2002 1:867-881.

Ribavirin (1a; 1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-1H-[1,2,4]triazole-3-carboxylic acid amide; Virazole) is a synthetic, non-interferon-inducing, broad spectrum antiviral nucleoside analog. Ribavirin has in vitro activity against several DNA and RNA viruses including Flaviviridae (Gary L. Davis, Gastroenterology 2000 118:S104-S114). In monotherapy ribavirin reduces serum amino transferase levels to normal in 40% of patients, but it does not lower serum levels of HCV-RNA. Ribavirin also exhibits significant toxicity and is known to induce anemia. Viramidine 1b is a prodrug converted to 1a in hepatocytes
Interferons (IFNs) have been available for the treatment of chronic hepatitis for nearly a decade. IFNs are glycoproteins produced by immune cells in response to viral infection. Two distinct types of interferon are recognized: Type 1 includes several interferon alphas and one interferon β, type 2 includes interferon γ. Type 1 interferons is produced mainly by infected cells and protects neighboring cells from de novo infection. IFNs inhibit viral replication of many viruses, including HCV, and when used as the sole treatment for hepatitis C infection, IFN suppresses serum HCV-RNA to undetectable levels. Additionally, IFN normalizes serum amino transferase levels. Unfortunately, the effects of IFN are temporary. Cessation of therapy results in a 70% relapse rate and only 10-15% exhibit a sustained virological response with normal serum alanine transferase levels. (L.-B. Davis, supra)
One limitation of early IFN therapy was rapid clearance of the protein from the blood. Chemical derivatization of IFN with polyethyleneglycol (PEG) has resulted in proteins with substantially improved pharmacokinetic properties. PEGASYS® is a conjugate interferon α-2a and a 40 kD branched mono-methoxy PEG and PEG-INTRON® is a conjugate of interferon α-2b and a 12 kD mono-methoxy PEG. (B. A. Luxon et al., Clin. Therap. 2002 24(9):13631383; A. Kozlowski and J. M. Harris, J. Control. Release, 2001 72:217-224).
Combination therapy of HCV with ribavirin and interferon-α currently represent the optimal therapy. Combining ribavirin and PEG-IFN (infra) results in a sustained viral response in 54-56% of patients. The SVR approaches 80% for type 2 and 3 HCV. (Walker, supra) Unfortunately, the combination also produces side effects which pose clinical challenges. Depression, flu-like symptoms and skin reactions are associated with subcutaneous IFN-α and hemolytic anemia is associated with sustained treatment with ribavirin.
A number of potential molecular targets for drug development as anti-HCV therapeutics have now been identified including, but not limited to, the NS2-NS3 autoprotease, the N3 protease, the N3 helicase and the NS5B polymerase. The RNA-dependent RNA polymerase is absolutely essential for replication of the single-stranded, positive sense, RNA genome. This enzyme has elicited significant interest among medicinal chemists.
Nucleoside inhibitors can act either as a chain terminator or as a competitive inhibitor which interferes with nucleotide binding to the polymerase. To function as a chain terminator the nucleoside analog must be taken up be the cell and converted in vivo to a triphosphate to compete for the polymerase nucleotide binding site. This conversion to the triphosphate is commonly mediated by cellular kinases which imparts additional structural requirements on a potential nucleoside polymerase inhibitor. In addition this limits the direct evaluation of nucleosides as inhibitors of HCV replication to cell-based assays capable of in situ phosphorylation.
In WO 01 90121 published Nov. 29, 2001, J.-P. Sommadossi and P. Lacolla disclose and exemplify the anti-HCV polymerase activity of 1′-alkyl- and 2′-alkyl nucleosides of formulae 2 and 3. In WO 01/92282, published Dec. 6, 2001, J.-P. Sommadossi and P. Lacolla disclose and exemplify treating Flaviviruses and Pestiviruses with 1′-alkyl- and 2′-alkyl nucleosides of formulae 2 and 3. In WO 03/026675 published Apr. 3, 2003, G. Gosselin discloses 4′-alkyl nucleosides 4 for treating Flaviviruses and Pestiviruses. In WO2004003000 published Jan. 8, 2004, J.-P. Sommadossi et al. disclose 2′- and 3′ prodrugs of 1′-, 2′-, 3′- and 4′-substituted β-D and β-L nucleosides. Idenix has reported clinical trials for a related compound NM283 which is believed to be the valine ester 5 of the cytidine analog 2 (B=cytosine).

In WO02/05787 published Jul. 25, 2002, S. S. Carroll et al. disclose related 2α-methyl and 2β-methylribose derivatives wherein the base is an optionally substituted 7H-pyrrolo[2,3-d]pyrimidine radical 6. The same application discloses one example of a 3β-methyl nucleoside. S. S. Carroll et al. (J. Biol. Chem. 2003 278(14):11979-11984) disclose inhibition of HCV polymerase by 2′-O-methylcytidine (6a).

Modification of nucleosides by substitution at the 4′-position has been less prevalent, most like due to the added synthetic challenges associated with their synthesis. Maag et al. (Anti-HIV Activity of 4′-Azido and 4′-Methoxynucleosides, J. Med. Chem. 1992 35:1440-1451) disclose the synthesis of 4′-azido-2-deoxyribonucleosides and 4-azido nucleosides. C. O'Yang, et al. (Tetrahedron Lett. 1992 33(1):37-40 and 33(1):41-44) disclose the synthesis 4′-cyano, 4′-hydroxymethyl- and 4′-formyl nucleoside compounds substituted nucleosides. These compounds were evaluated as anti-HIV compounds.
In WO02/100415 published Dec. 19, 2002 (US 2003/0236216 A1), R. R. Devos et al. disclose 4′-substituted nucleoside compounds that exhibit HCV activity. Four compounds explicitly identified include the 4′-azido compound, 7a, the 4′-ethynyl compound 7b, the 4′-ethoxy compound 7c and the 4′-acetyl compound 7d. Modifications to the ribose moiety exemplified include the 2′-deoxy 8a derivative, 3′-deoxy derivative 8b, the 3′-methoxy derivative 8e, the 3′-fluoro derivative 8c and the 2′,2′-difluoro derivative 8d. In WO2004/046159 published Jun. 3, 2004 (US 2004121980), J. A. Martin et al. disclose prodrugs of 7a useful for treating HCV-mediated diseases. Both US applications are hereby incorporated by reference in their entirety. While compounds with the arabinose configuration fall within genus, these compounds are not among specifically disclosed, exemplified, or included in the preferred list of nucleosides in the specification.

U.S. application Ser. No. 10/167,106 filed Jun. 11, 2002 entitled “4′-Substituted Nucleoside Derivatives as Inhibitors of HCV RNA Replication”, and U.S. application Ser. No. 10/717,260 file Nov. 19, 2003 disclose compounds related to the present invention. Both applications are incorporated herein in their entirety by reference.
Y.-H. Yun et al. (Arch. Pharm. Res. 1985 18(5):364-35) disclose the synthesis and antiviral activity of 4′-azido-2′-deoxy-2′-fluoro-arabinofuranosyl nucleosides (9: R=H, Me and Cl).

G. S. Jeon and V. Nair (Tetrahedron 1996 52(39): 12643-50) disclose the synthesis 4′-azidomethyl-2′, 3′-deoxyribonucleosides 10 (B=adenine, thymine and uracil) as HIV reverse transcriptase inhibitors.
Several computational studies of 4′-azidonucleosides have been reported: D, Galisteo et al., J. Mol. Struct. 1996 384(1):25-33; J. Pepe et al., Eur. J. Med. Chem. 1996 32(10):775-786; E. Estrada et al., In silico studies toward the discovery of New Anti HIV Nucleoside, J. Chem. Info. Comp. Sci. 2002 42(5): 1194-1203;
I. Sugimoto et al. disclosed the synthesis and the HIV and H. simplex bioassay of 4′-ethynyl-2′-deoxycytidine (11) and other two-carbon substituents at the 4′-position (Nucleosides and Nucleotides. 183. Synthesis of 4′ α-Branched Thymidines as a New Type of Antiviral Agent, Bioorg Med. Chem. Lett. 1999 9:385-88). T. Wada et al. (Nucleosides & Nucleotides 1996 15(1-3):287-304) disclose the synthesis and anti-HIV activity of 4′-C-methyl nucleosides.
In WO 01/32153 published May 10, 2001, R. Storer discloses methods of treating or preventing Flaviviridae viral infection by administering dioxolane analogs of nucleosides
In WO02/18404 published Mar. 7, 2002, R. Devos et al. disclose novel and known purine and pyrimidine nucleoside derivatives and their use as inhibitors of subgenomic HCV replication and pharmaceutical compositions containing said nucleoside derivatives. The compounds disclosed consist of nucleosides with substituted purine and pyrimidine bases.
Several references have reported the synthesis and use of fluoro-nucleosides with the arabinose configuration for therapy of viral diseases. There have been several reports of 2-fluoro-β-D-arabinofuranosyl nucleosides that exhibit activity against hepatitis B and herpes. See, for example, U.S. Pat. No. 6,348,587 B1 (R. F. Schinazi et al.), U.S. Pat. No. 4,666,892 (Fox, et al.); U.S. Pat. No. 4,211,773 (Lopez, et al); Su, et al., Nucleosides. 136, Synthesis and Antiviral Effects of Several 1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)-5-alkyluracils. Some Structure-Activity Relationships, J. Med. Chem. 1986 29:151-154; Borthwick, et al., Synthesis and Enzymatic Resolution of Carbocyclic 2′-Ara-fluoro-Guanosine: A Potent New Anti-Herpetic Agent, J. Chem. Soc., Chem. Commun. 1988; Wantanabe, et al., Synthesis and Anti-HIV Activity of 2′-“Up”-Fluoro Analogues of Active Anti-Aids Nucleosides 3′-Azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxycytidine (DDC), J. Med. Chem. 1990 33:2145-2150; Martin, et al., Synthesis and Antiviral Activity of Monofluoro and Difluoro Analogues of Pyrimidine Deoxyribonucleosides against Human Immunodeficiency Virus (HIV-1), J. Med. Chem. 1990 33:2137-2145; Sterzycki et al., Synthesis and Anti-HIV Activity of Several 2′-Fluoro-Containing Pyrimidine Nucleosides, J. Med. Chem. 1990; and Montgomery, et al., 9-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)guanine: A Metabolically Stable Cytotoxic Analogue of 2′-Deoxyguanosine. U.S. Pat. No. 5,246,924 discloses a method for treating a hepatitis infection that includes the administration of 1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-3-ethyluracil). U.S. Pat. No. 5,034,518 discloses 2-fluoro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine nucleosides which exhibit anticancer activity by altering the metabolism of adenine nucleosides by reducing the ability of the compound to serve as a substrate for adenosine. EPA 0 292 023 discloses that certain β-D-2′-fluoroarabinonucleosides are active against viral infections
It has also been disclosed that L-FMAU (2′-fluoro-5-methyl-β-L-arabinofuranosyluracil) is a potent anti-HBV and anti-EBV agent. See Chu, et al., Use of 2′-Fluoro-5-methyl-β-L-arabinofuranosyluracil as a Novel Antiviral Agent for Hepatitis B Virus and Epstein-Barr Virus Antimicrobial Agents and Chemotherapy, 1995 39(4):979-98; Balakrishna, et al., Inhibition of Hepatitis B Virus by a Novel L-Nucleoside, 2′-Fluoro-5-Methyl-β-L-arabinofuranosyl Uracil, Antimicrobial Agents and Chemotherapy, 1996 40(2):380-356; U.S. Pat. Nos. 5,587,362; 5,567,688; and 5,565,438.
EPA Publication No. 0 352 248 discloses a broad genus of L-ribofuranosyl purine nucleosides for the treatment of HIV, herpes, and hepatitis. A similar specification is found in WO 88/09001, filed by Aktiebolaget Astra.
European Patent Application 0 357 571 discloses a broad group of β-D and α-D pyrimidine nucleosides for the treatment of AIDS which among the broad class generically includes nucleosides that can be substituted in the 2′ or 3′-position with a fluorine group.

H. Ohrui et al. (Antimicrobial Agents and Chemother. 2001 45(5):1539-1546; see also S. Koghgo et al., Tennen Yuki Kagobutsu Toronkai Koen Yoshishu 2000 42:835 (Chem. Abs. 2001:102156 anad H. Ohrui et al. WO2000069876 published Nov. 23, 2000) disclose the synthesis and anti-HIV activity of 4′-C-ethynyl-β-D-arabino- and 4′-C-ethynyl-2′-deoxy-β-D-ribo-pentofuranosyl pyrimidines and -purines. 4-Ethynyl-cytarabine (12a) exhibits good anti-HIV activity while the corresponding nucleoside wherein the base was thymine 12b was inactive. Several 4′-C-ethylnyl-2′-deoxy-β-D-ribo-pentofuranosyl pyrimidines and -purines were potent inhibitors of HIV reverse transcriptase (HIV-RT).
K. Kitano et al. (Tetrahedron 1997 53(39): 13315-13322) disclose the synthesis 4′-fluoromethyl 2-deoxy-D-erythro-, ribo- and arabino-pentofuranosyl cytosines and anti-neoplastic activity.
Intensive effort has focused on the identification of non-nucleoside inhibitors of HCV NS5B polymerase. The results of these efforts have been reviewed (J. Z. Chen and Z. Hong, Targeting NS5B RNA-Dependent RNA Polymerase for Anti-HCV Chemotherapy, Curr. Drug Targ. Inf Dis. 2003 3(3):207-219). The non-nucleoside inhibitors are not related to the present invention.
The object of the present invention is to provide new compounds, methods and compositions for the treatment of a host infected with hepatitis C virus.